Epithelial to mesenchymal transformation in tubular epithelial cells undergoing anoxia.
OBJECTIVE
Epithelial-mesenchymal transformation (EMT) has been proved to be a critical event in fibrogenesis of renal allografts. This study sought to determine whether anoxia could induce EMT from tubular epithelial cells (TEC).


METHODS
Rat TEC-line (NRK-52E) was cultured in Dulbelco's modified Eagle's medium (DMEM) without glucose under 100% N2 for 4 hours. After 6, 12, 24, 48, and 72 hours, the expressions of connective tissue growth factor (CTGF) mRNA and protein were measured by real-time RT-PCR and Western blot, respectively. Morphologic changes and cytoskeleton remodeling were observed in NRK-52E cells under laser confocal microscopy. Immunohistochemistry and flow cytometry were used to detect expression changes of E-cadherin, alpha-smooth muscle actin (SMA), types I and IV collagen, all of which are involved in TEC, EMT.


RESULTS
After stimulation by anoxia, NRK-52E cells became round and enlarged with a remodeled cytoskeleton. The expressions of CTGF mRNA and protein were upregulated after 6 hours, reaching their peak at 48 hours. The expressions of types I and IV collagen, and alpha-SMA were all upregulated except for E-cadherin.


CONCLUSIONS
Anoxia upregulated the expression of CTGF and other EMT-associated genes in NRK-52E cells.
Luo
G H
GH
Transplantation Institute, West China Hospital, Sichuan University, Chengdu, China.
Lu
Y P
YP
Yang
L
L
Song
J
J
Shi
Y J
YJ
Li
Y P
YP
D000199:Actins / Q000235:genetics; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics; D024141:Collagen Type IV / Q000235:genetics; D055513:Connective Tissue Growth Factor / Q000235:genetics; D017931:DNA Primers; D004847:Epithelial Cells / Q000166:cytology* / Q000502:physiology; D005786:Gene Expression Regulation; D018969:Insulin-Like Growth Factor Binding Proteins / Q000235:genetics; D007668:Kidney; D008648:Mesoderm / Q000166:cytology* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D000199:Actins; D024042:Collagen Type I; D024141:Collagen Type IV; D055513:Connective Tissue Growth Factor; D017931:DNA Primers; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; C498862:Acta2 protein, rat